Gene silencing, i.e., selectively blocking the expression of a gene of interest, may be effected via the introduction of an antisense oligonucleotide (AON) or small interfering RNA (siRNA) into an organism (Uhlmann, E. and Peyman, A. Chem. Rev. 1990, 90: 543-84; Braasch, D. A. and Corey, D. R. Biochemistry 2002, 41: 4503-4510; Opalinska, J. B. and Gewirtz, A. M. Nat. Rev. Drug Discov. 2002, 1: 503-14; Dorsett, Y. and Tuschl, T. Nat. Rev. Drug Discov. 2004, 3: 318-329). Unfortunately, as with other nucleic acid-based drugs, siRNAs have poor serum stability, poor cellular uptake, and can elicit off-target and immunostimulatory side effects. Efforts to remedy these shortcomings have focused on the development of delivery vehicles for siRNAs, and on the development of chemically modified oligonucleotides with improved drug profiles.
Much recent work has focussed on the chemical modification of siRNA. Dowler et al. (Dowler, T. et al. Nucl. Acids Res. 2006, 34: 1669-1675) were the first to show that 2′-deoxy-2′F-arabinonucleic acids (2′F-ANA) could be incorporated throughout the sense strand, including a fully-modified sense strand. Modification of the antisense-strand 3′-overhang with 2′F-ANA brought a significant increase in potency, and several of the 2′F-ANA-modified duplexes have been able to surpass the native siRNA in potency. Furthermore, siRNA duplexes with extensive 2′F-ANA modification were found to have a significantly longer serum half-life than unmodified siRNAs. Modified siRNA duplexes containing 2′-fluoro-4′-thioarabinonucleotide (4′S-FANA) units were able to enter the RNAi pathway (Watts, J. K. et al. Nucl. Acids Res. 2007, 35: 1441-1451). One or two inserts internally in either strand gave duplexes of potency comparable to that of the control. The 4′S-FANA modification was also able to work with good efficiency in a duplex with a modified 2′F-ANA-RNA sense strand, demonstrating that 2′F-ANA (with its preference for southern and eastern conformations) can achieve synergy with 4′S-2′F-ANA (with its preference for northern conformations), in RNAi gene silencing.
2′F-RNA is another siRNA modification, and partial 2′F-RNA modification is tolerated throughout both the sense and antisense strands, and some fully-modified 2′F-RNA siRNAs are also active. 2′F-RNA-modified siRNA duplexes have significantly increased serum stability (Layzer, J. M. et al. RNA, 2004, 10: 766-771). 2′F-RNA also increases the binding affinity of the duplex.
An example of an increase in potency was observed for a fully modified siRNA made of a combination of 2′-O-Me and 2′F-RNA modified nucleotides, which was 500 times more potent than unmodified RNA (Allerson, C. R. et al. J. Med. Chem. 2005, 48: 901-904; Koller, E. et al. Nucl. Acids Res. 2006 34: 4467-4476). However, such a high degree of improvement was not observed for other sequences.
These techniques present significant challenges, and there is a need for improvements in for example efficacy, in vivo stability and reduction of “off-target” effects (e.g., the silencing of a gene other than the intended target). There is therefore a continued need for improved oligonucleotide-based approaches.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.